This project is designed to define the nature of interaction between tRNA and aminoacyl-tRNA synthetases. Chemical and enzymatic modifications of arginine and lysine tRNA's will be carried out to produce identified alterations in specific bases or in specific phosphodiester bonds. The effects of these alterations on the interaction of these nucleic acids with cognate enzymes will be evaluated by kinetic analyses. Since kinetic analyses depend upon the mechanism of enzyme action, studies will also be continued to determine the mechanism of action of the synthetases. These include kinetic analyses of partial reactions in which chemically synthesized aminoacyl adenylates serve as substrates for pyrophosphorolysis and transfer to tRNA. An attempt will also be made to terminate the controversy about the possibility of a concerted mechanism by showing that the intermediary complex does transfer amino acid at least as fast as the overall reaction, and that experiments that purport to show otherwise are influenced by a technical artifact in which an exchange reaction equilibrates amino acid in the complex with the pool of free amino acid. Additional experiments will also be designed to identify specific amino acid residues in the enzymes that participate in binding and catalysis. BIBLIOGRAPHIC REFERENCES: Kalpana Chakraburtty, Christian F. Midelfort, Assaf Steinschneider and Alan H. Mehler (1975) The Role of Divalent Cations in the Reactions of Valyl Transfer Ribonucleic Acid Synthetase of Escherichia coli, J. Biol. Chem. 250:3861-3865. Christian F. Midelfort, Kalpana Chakraburtty, Assaf Steinschneider and Alan H. Mehler (1975) Kinetic Demonstration of the Intermediate Role of Aminoacyl-Adenylate Enzyme in the Formation of Valyl Transfer Ribonucleic Acid, J. Biol. Chem. 250 3866-3873.